Suppression of DNA-Damage Checkpoint Signaling by Rsk-Mediated Phosphorylation of Mre11

Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11

Chen Chen, Liguo Zhang, Nai-Jia Huang, Bofu Huang, and Sally Kornbluth1

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27710

Edited by Tony Hunter, The Salk Institute for Biological Studies, La Jolla, CA, and approved November 12, 2013 (received for review April 9, 2013)

Ataxia telangiectasia mutant (ATM) is an S/T-Q–directed kinase A hallmark of cancer cells is their ability to override cell-cycle that is critical for the cellular response to double-stranded breaks checkpoints, including the DSB checkpoint, which arrests the cell (DSBs) in DNA. Following DNA damage, ATM is activated and cycle to allow adequate time for damage repair. Previous studies recruited by the MRN protein complex [meiotic recombination 11 have implicated the MAPK pathway in inhibition of DNA-dam- (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome age signaling: PKC suppresses DSB-induced G2/M checkpoint 1 proteins] to sites of DNA damage where ATM phosphorylates signaling following ionizing radiation via activation of ERK1/2 multiple substrates to trigger cell-cycle arrest. In cancer cells, this (22); activation of RAF kinase, leading to activation of MEK/ regulation may be faulty, and cell division may proceed even in ERK/Rsk, also can suppress G2/M checkpoint signaling (23). the presence of damaged DNA. We show here that the ribosomal Given its prominent role in multiple cancers, the MAPK s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced pathway is an attractive therapeutic target. Indeed, treatment of melanoma using the RAF inhibitor vemurafenib has shown some ATM activation in both Xenopus egg extracts and human tumor cell clinical success, as has treatment of nonsmall cell lung carcinoma lines. In analyzing each step in ATM activation, we have found that with MEK inhibitors (24). However, targeting components at the Rsk targets loading of MRN complex components onto DNA at DSB apex of a signaling pathway may induce side effects caused by the sites. Rsk can phosphorylate the Mre11 protein directly at S676 both plethora of downstream effectors (25, 26). As a terminal kinase in vitro and in intact cells and thereby can inhibit the binding of in the MAPK pathway, Rsk may avoid these complications as Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can a potential target. Thus, there has been interest in targeting Rsk reverse inhibition of the response to DSBs by Rsk. Collectively, these for cancers with notable Rsk elevation (e.g., prostate cancers) (27). data point to Mre11 as an important locus of Rsk-mediated check- Several Rsk-specific inhibitors have been described, including point inhibition acting upstream of ATM activation. SL0101 and BI-D1870 (18, 28, 29). Whether these or derivative drugs will be clinically successful remains unclear. However, if Rsk dsDNA IP | PMA | SL0101 | γ-H2AX inhibition can reinstate DSB-induced checkpoint function, then combination therapy of Rsk inhibitors with DNA-damaging agents ells have evolved multiple pathways signaling DNA damage may be effective in inducing tumor cell arrest or death. Cthat trigger DNA repair, cell-cycle arrest and, in the event The precise mechanism underlying checkpoint inhibition down- of irreparable damage, cell death. Among the various forms of stream of MEK/ERK/Rsk signaling is not yet clear. One recent DNA damage, double-stranded breaks (DSBs) in DNA, gener- paper has shown that, after doxorubicin-induced DNA damage (both DSB and ssDNA breaks), Rsk can silence the G2/M ated by exposure to ionizing radiation and radiomimetic chemicals checkpoint by phosphorylating (and inhibiting) the Chk1 kinase such as neocarzinostatin (NCS), are the most lethal for cells (1). on S280 (the same site that can be targeted by protein kinase B/ DSBs often are repaired by homologous recombination during Akt kinase) (30). However, another group has reported that the S and G2/M phases of the cell cycle, which involves the phosphorylation of this site on Chk1 enhanced its ability to en- meiotic recombination 11 (Mre11)/DNA repair protein Rad50/ force the checkpoint by promoting its nuclear translocation (31). Nijmegen breakage syndrome 1 (Nbs1) (MRN) complex and the We show here that Rsk signaling silences the DSB-induced ataxia telangiectasia mutated (ATM) kinase (2, 3). The MRN fi G2/M checkpoint by preventing activation of the ATM kinase. complex rst recognizes sites of DNA damage and then pro- Specifically, we have found that Rsk targets the Mre11 compo- motes binding of ATM to the DSB site. ATM, activated by nent of the MRN complex by phosphorylating S676, thereby monomerization and autophosphorylation, phosphorylates down- preventing Mre11 binding to DNA. Mutation of S676 restored stream proteins including p53, checkpoint kinase 2 (Chk2), and ATM activation, even in the face of high Rsk activity, and rendered breast cancer 1, early onset (BRCA1) (4-7). These factors then convey the signal to induce cycle arrest, apoptosis, or DNA repair Significance (8). Failure of this process results in genome instability, increasing

the risk of cancer, neurodegeneration, and other pathologies (9). CELL BIOLOGY Ribosomal S6 kinase (Rsk), which functions downstream of We have identified a pathway that links MAPK/ribosomal s6 mitogen-activated protein kinase kinase (MEK) and ERK, is kinase (Rsk) signaling to the inhibition of the response to frequently activated in cancer cells (10, 11). Rsk activation can a double-stranded break (DSB) in DNA, potentially explaining be promoted by multiple signaling pathways in cancer cells, in- radioresistance in tumors with high rapidly accelerated fibro- cluding those triggered by steroids, insulin, EGF, and estrogen sarcoma (Raf)/MAPK/Rsk activity. The locus of this regulation (10, 12–15). Additionally, Rsk activation can be triggered by was identified as meiotic recombination 11, whose phosphor- PKC signaling [via the PKC/rapidly accelerated fibrosarcoma ylation by Rsk prevented its DNA binding and consequent ac- (RAF)/mitogen-activated protein kinases (MAPK) pathway], tivation of the central DSB checkpoint regulator, ataxia which is activated by phorbol12-myristate13-acetate (PMA) (16, telangiectasia mutant. 17). Previous studies have found that Rsk2 is overexpressed in 50% of breast cancers and prostate tumors (18, 19), and Rsk Author contributions: C.C. and S.K. designed research; C.C., L.Z., N.-J.H., and B.H. per- signaling has been implicated in the regulation of survival, an- formed research; C.C. analyzed data; and C.C. and S.K. wrote the paper. chorage-independent growth, and transformation of breast cancer The authors declare no conflict of interest. cells in culture (20). Rsk-specific inhibition (with BI-D1870 or This article is a PNAS Direct Submission. SL0101) significantly reduced proliferation of MCF7, PC3, or 1To whom correspondence should be addressed. E-mail: [email protected]. LnCaP cancer cells (18, 19). Rsk also inhibits apoptosis in PC3 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. prostate cancer cells (21). 1073/pnas.1306328110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306328110 PNAS | December 17, 2013 | vol. 110 | no. 51 | 20605–20610 Downloaded by guest on October 2, 2021 cells refractory to the checkpoint-inhibitory effects of Rsk. This suggest that Rsk might inhibit the DSB checkpoint in PC3 prostate finding is consistent with a previous study showing that phosphor- cancer cells. To examine this possibility further, we stained PC3 ylation on Mre11 is mostly inhibitory (32). Taken together, these cells for γ-H2AX, a downstream target of ATM. As shown in Fig. findings identify a locus of checkpoint regulation by Rsk and 1B, PC3 cells treated with SL0101 before NCS treatment dis- support the idea of targeting Rsk for therapeutic benefit. played greatly increased γH2AX staining. These data strongly suggest that high Rsk activity can suppress ATM-dependent G2/M Results checkpoint signaling. Prostate Cancer Cells with High Levels of Endogenous Rsk Are Resistant Although it has been reported that inhibiting ATM may en- hance G2/M accumulation (33), inhibiting ATM decreased the to DSBs. Rsk is highly expressed in many prostate cancer cell lines, B including PC3 and LnCaP cells, which appear to be highly de- G2/M population in the cell lines we used (Fig S1 ). This result pendent on Rsk activity for growth and survival (19). We hy- also is consistent with other published studies (34, 35). To determine if Rsk is sufficient to confer resistance to DSB- pothesized that Rsk might modulate the response of these cells induced G2/M arrest, we overexpressed constitutively active Rsk2 to DNA damage-induced checkpoint function. To evaluate this in 293T cells, which have much lower endogenous levels of Rsk hypothesis, we analyzed cell-cycle progression after DSB induction than PC3 cells. As shown in Fig. 1C, Rsk-expressing cells were in PC3 cells that endogenously express twice the level of Rsk considerably less sensitive to DNA damage than control cells. To protein found in normal prostate tissue (19). Cells were arrested at demonstrate G2/M checkpoint suppression, directly, we synchro- G1/S using double thymidine block and 4 h after release were nized HeLa cells in G1/S phase by double thymidine block and treated with PMA and/or SL0101, followed by NCS to induce treated them 4 h after release with PMA or SL0101 for 30 min and DSBs during the G2 phase. After drugs were washed away, cells with NCS for an additional 30 min. Three or five hours later, cells were incubated in fresh media for 16 h before DNA contents were were stained with anti–phospho-Histone H3 (PHH3) antibody. As analyzed. As shown in Fig. 1A, cotreatment of cells with SL0101 shown in Fig. S1C, NCS significantly reduced the number of pHH3- and NCS resulted in much more robust G2/M arrest than seen positive cells by arresting cells in G2 phase. However, PMA treat- with NCS alone. Additionally, after PMA-induced Rsk activation, ment greatly increased the pHH3-positive population (particularly the DSB-induced G2/M arrest was effectively overridden. We at 5 h), suggesting that the G2/M checkpoint had been silenced. obtained similar results in experiments in which Rsk1/2 were This checkpoint inhibition was reversed by cotreatment with knocked down using siRNA (Fig S1A). Taken together, these data SL0101, confirming a role for Rsk in this silencing.

A 70 * * * G2/M%

40 Ctrl PMA SL0101

pS6 37 kDa

50 kDa actin 37 kDa 0

Ctrl NCS Ctrl+PMA NCS+PMA Ctrl+SL0101 NCS+SL0101 Ctrl+PMA+SL0101 NCS+PMA+SL0101

B gamma-H2AX Hoechst Merged

NCS average 700 gamma-H2AX ** intensity 600

* 400 Fig. 1. Rsk suppresses the DSB response in prostate NCS + cancer. (A) PC3 cells were synchronized in G1/S SL0101 200 phase by double thymidine block. Four hours after release, cells were treated with PMA or SL0101 as 0 indicated and then with NCS and were analyzed for NCS ONLY SL0101 SL0101 DNA content. n = 3 experiments. The effectiveness SL0101 +NCS of PMA and SL0101 in modulating Rsk activity was shown by immunoblotting for the Rsk substrate, S6. *P < 0.01; **P < 0.01. Error bars indicate SD. (B)PC3 cells grown on coverslips were treated with SL0101 C G2/M G2/M percentage% or DMSO and then with NCS. Fixed cells stained for 60 * NS NS γH2AX were examined by fluorescent microscopy (20×). The average intensity of γH2ax staining for more than 50 cells from at least seven random fields 40 was quantified with MetaMorph. *P < 0.01; **P < 0.01. (C) 293T cells transfected with FLAG-tagged 20 WT or constitutively active Rsk2 were treated with NCS. DNA profiles of FLAG-positive sorted cells = 0 were analyzed to quantify cells in G2/M. n 3 Ctrl-NCS Ctrl+NCS RskWT RskWT RskCA RskCA experiments. *P < 0.05. Error bars indicate SD. P -NCS +NCS -NCS +NCS values are derived from the Student t test.

20606 | www.pnas.org/cgi/doi/10.1073/pnas.1306328110 Chen et al. Downloaded by guest on October 2, 2021 Rsk Suppresses the DSB Response by Inhibiting the Activation of ATM. - - + + Rsk2 To identify the locus of Rsk action within the DSB-signaling A pathway, we assessed the status of the ATM kinase following - + - + NCS NCS treatment in cells overexpressing constitutively active Rsk2. We found that after treatment with NCS these cells had reduced pATM 1981 250 kDa ATM phosphorylation at S1981 (a hallmark of ATM activation) A but no change in ATM protein levels (Fig. 2 ). Similarly, PMA ATM treatment markedly decreased ATM autophosphorylation at 250 kDa

S1981. However, ATM phosphorylation was restored when cells 100 kDa were treated with both PMA and SL0101 (Fig. S2A). To dem- FLAG-Rsk2 onstrate further the down-regulation of ATM, we measured phosphorylation of NBS1 at S343, a site known to be targeted by ATM. As expected, phosphorylation of NBS1 was reduced in the B Ctr SiRNA Rsk1/2 siRNA presence of PMA, consistent with Rsk-mediated reduction in NCS - + - + Ctr siRNARsk1/2 siRNA ATM activity (Fig. S2B). Accordingly, siRNA knock down of Rsk ATM 1981P Rsk1 85 kDa in PC3 cells significantly increased ATM autophosphorylation in 250 kDa response to NCS treatment (Fig. 2B). ATM autophosphorylation ATM Rsk2 85 kDa is a very early event in the DSB response, and these data suggest 250 kDa that Rsk acts either at or upstream of ATM. As is consistent with Actin 46 kDa pS6 an effect of Rsk signaling on ATM activity, HeLa cells treated 30 kDa with PMA stained more weakly with antibody directed against C D γ gamma-H2AX intensity H2AX than did untreated cells, and cotreatment with PMA and average relative to non-transfected SL0101 reversed the diminution in γH2AX staining (Fig. 2C and gamma-H2AX C intensity * Fig. S2 ). HeLa cells transfected with FLAG-tagged constitu- * ** tively activated Rsk2 exhibited significantly less γH2AX staining 100 1.4 than nontransfected cells after NCS treatment (Fig. 2D and Fig. D D S2 ). This effect was reversed by treatment with SL0101 (Fig. 2 1 and Fig. S2D). Collectively, these data suggest that Rsk signaling 50 suppresses both ATM activation and phosphorylation of its downstream targets, NBS1 and H2AX. 0.6 It is worth noting that ATM activation is detected robustly only 0 at the early phases of the DSB response (and thus the effects of Control PMA PMA+SL0101 Rsk could not be detected later, at 24 h) (Fig. S2E). This situation 0 non- Flag-Rsk2 Flag-Rsk2 probably reflects checkpoint adaptation or recovery from DNA transfected + SL0101 damage, as is consistent with previous reports (36, 37). However, cells were still phenotypically arrested at 24 h because either (i) fi Fig. 2. Rsk suppresses the DSB response by inhibiting the activation of ATM. the remaining ATM activity/DSB response is suf cient to maintain (A) HeLa cells transfected with FLAG-tagged Rsk2 were treated with NCS, the checkpoint or (ii) checkpoint recovery lags behind DSB re- lysed, and immunoblotted for phospho-ATM 1981. (B) Rsk1/2 knockdown sponse recovery because cells require time to resume the cell cycle PC3 cells were lysed and immunoblotted for phospho-ATM 1981. Cells even though the upstream DNA-damage signals have waned. treated with control siRNA were used as control. (C) HeLa cells grown on coverslips and treated with PMA ± SL0101 and then with NCS were fixed, The Moloney Sarcoma Oncogene/MAPK/Rsk Pathway Inhibits Binding stained, and examined for DAPI and γH2AX staining by fluorescence mi- of the MRN Complex to dsDNA. To identify the direct targets of Rsk croscopy (20×). Average intensity of γH2ax staining from more than 50 cells responsible for inhibiting the DSB response, we used the Xen- from 10 random fields was quantified with MetaMorph. *P < 0.01; **P < opus egg extract system, in which DSB checkpoint signaling can 0.01. Error bars indicate SD. (D) HeLa cells, transfected with FLAG-tagged γ be reconstituted in vitro. This system allows easy manipulation of constitutively active Rsk2 were examined for H2ax staining in FLAG-positive and -negative cells by fluorescence microscopy (40×). Average intensity of both DNA templates and Rsk signal components. Interphase γ – extracts prepared from Xenopus eggs (crude S extracts) were H2ax staining from more than 40 FLAG-Rsk positive and more than 300 FLAG-Rsk–negative cells from at least 18 random fields was quantified with treated with recombinant maltose-binding protein (MBP)-tagged MetaMorph. Fold change in intensity of γH2ax staining in FLAG-Rsk–positive Moloney sarcoma oncogene (Mos) kinase, a strong upstream relative to –negative cells was calculated. *P < 0.01. Error bars indicate SEM. activator of the MAPK pathway, leading to robust Rsk activation Statistical analyses were done by the Student t test. Representative images (38). This treatment markedly inhibited autophosphorylation of from C and D are shown in Fig. S2 C and D, respectively. ATM induced by dsDNA without altering ATM levels. SL0101 treatment reversed this inhibition (Fig. 3A). To rule out the CELL BIOLOGY possibility that signaling by other Mos-activated kinases might in ATM/DNA binding (Fig. 3D). Together, these data suggest that cause the inhibition of the DSB response, we treated crude S Rsk functions upstream of ATM, affecting its recruitment to sites extracts with prephosphorylated constitutively activated Rsk, and of DNA damage. observed a similar decrease in ATM autophosphorylation (Fig. Because ATM recruitment is preceded by recognition of DNA- B 3 ). Because ATM must be recruited to the site of DNA break damage sites by the MRN complex, we investigated whether the by the MRN complexes before autophosphorylation and mono- binding of the MRN complex to dsDNA is affected by Rsk. In- merization, we investigated the binding of ATM to dsDNA. We deed, less Mre11 protein was associated with dsDNA in the crude immobilized biotin-tagged dsDNA on avidin beads to mimic sites S extracts pretreated with Mos than with control MBP protein. of dsDNA damage, incubated the beads with crude S extracts, D and retrieved DNA bead-associated proteins. We observed Moreover, SL0101 suppressed the effect of Mos (Fig. 3 ). The a significant decrease in dsDNA-bound ATM after pretreatment association of Mre11 with the beads was DNA dependent; an fi of extracts with Mos, whereas binding of another DNA damage- excess of free DNA signi cantly decreased the levels of bead- response protein, Ku70, was unchanged (Fig. 3C). To rule out bound Mre11 (Fig. S3A). Moreover, treatment with either the the possibility that decreased ATM binding resulted from re- ATM inhibitor KU55933 or ATM/ATR inhibitor caffeine did not duced ATM activity, we supplemented extracts with the ATM/ reverse Rsk’s inhibition of Mre11 binding. This result strengthens ataxia telangiectasia and Rad3-related protein (ATR) kinase in- the suggestion that the observed effect on MRN complex/DSB hibitor caffeine; this supplementation did not reverse the decrease DNA assembly was not caused by feedback from downstream

Chen et al. PNAS | December 17, 2013 | vol. 110 | no. 51 | 20607 Downloaded by guest on October 2, 2021 (dAdT) 70 using the recombinant mutants. Although several of the sites A B -Rsk +Rsk Mos SL0101 MBP Mos 0’ 10’ 20’ 30’ 0’ 10’ 20’ 30’ reduced phosphorylation, the single S676A mutation abolished +SL0101 phosphorylation almost completely, suggesting that S676 is the 0’10’ 20’ 30’ 0’10’ 20’ 30’ 0’ 20’10’ 30’ 0’ 20’10’ 30’ phospho-ATM 250 kDa major site of in vitro phosphorylation by Rsk (Fig. 4C). Mre11 phospho-ATM 250 kDa phosphorylation also was severely compromised by mutation of ATM ATM 250 kDa 250 kDa S676 when radiolabeled ATP was added to lysates that had been prepared from cells transfected with Rsk (Fig. S4B). Note that in Empty No Caffeine +Caffeine Empty Beads C Beads No Caffeine +Caffeine D - ++ - + Mos this experiment, cells were not serum starved before lysate pro- - - - - + - + Mos - + - - SL0101 duction, and thus WT or constitutively activated Rsk proteins

ATM 250 kDa Mre11 75 kDa behaved similarly (because of the activation of the transfected WT Rsk). As is consistent with our in vitro data, we successfully 75 kDa 75 kDa KU70 KU70 detected endogenous Mre11 S676 phosphorylation in the presence of PMA or overexpressed WT/constitutively active Rsk with Fig. 3. The Mos/MAPK/Rsk pathway inhibits binding of the MRN complex to an antibody that targeted phospho-S676 (Fig. 4 D and E). Phos- dsDNA. (A) Xenopus crude interphase extracts (S extracts) were treated with phorylation induced by PMA was abrogated by cotreatment with control MBP or MBP-Mos and then with SL0101 or DMSO, and double- the Rsk inhibitor SL0101 (Fig. 4 D and E). In addition, we treated stranded poly (dA-dT)70. Samples were analyzed for ATM autophosphor- Rsk-transfected cells with a DNA PK inhibitor to rule out the ylation by immunoblotting. (B) Xenopus crude S extracts were incubated possibility that Rsk might cause Mre11 S676 phosphorylation in- with active RSK for 15 min before the addition of dsDNA. Samples were directly by activating DNA PK (Fig. S5A). Although the DNA PK analyzed for ATM autophosphorylation by immunoblotting. (C) Crude S inhibitor was effective against a known DNA PK substrate, Akt, it extracts were pretreated with MBP-Mos or MBP alone and caffeine and were did not inhibit phosphorylation of Mre11 downstream of Rsk (Fig. incubated with biotin-tagged dsDNA-bound avidin beads. The amount of B ATM bound to beads was analyzed by immunoblotting. DNA bound Ku70 S5 ). Collectively, these data suggested that Rsk can phosphory- was used as loading control. (D) Xenopus crude S extracts were pretreated with late S676 of Mre11 both in vitro and in intact cells. MBP-Mos or MBP ± caffeine or SL0101 and were incubated with biotin-tagged dsDNA which was bound to avidin beads. The amount of Mre11 protein bound Phosphorylation of Mre11 on S676 Reduces Its Binding to dsDNA and to beads was analyzed by immunoblotting. Ku70 was the loading control. Is Responsible for Checkpoint Inhibition by Rsk. Mre11 has two potential DBDs, and S676 is located within the second of these (Fig. 5A) (41, 42). Therefore, we speculate that phosphorylation of ATM signaling (Fig. 3D and Fig. S3A). To confirm these findings Mre11 on S676 might directly disrupt its binding to dsDNA. As in intact cells, we stained HeLa cells with anti-Mre11 antibody. shown in Fig. 5B, Mos treatment of egg extracts reduced binding PMA activation of Rsk significantly reduced the formation of of WT Mre11 to DNA beads but had no effect on the S676A Mre11/MRN foci, suggesting that Rsk indeed was suppressing the mutant. Moreover, pretreating recombinant WT Mre11 with Rsk binding of the MRN complex to the DSB DNA (Fig. S3B). To rule out the possibility that Rsk disrupts the interaction between Mre11 and other MRN complex components, we in- A B vestigated binding of Mre11 to Nijmegen breakage syndrome 1 WT Mre11 - Control Rsk2 Rsk2* Input ++ Empty beads proteins (Nbs1) in egg extracts without added DNA. We did not Rsk2 + - + observe any effect of Rsk on the phys-ical interaction between 100 kDa 100 kDa C 32P Mre11 these two proteins (Fig. S3 ), supporting the hypothesis that Rsk 75 kDa Mre11 75kDa interferes with Mre11 binding to DNA but not with the forma- 100 kDa fi Loading: Mre11 tion of the MRN complex itself. To con rm that Rsk mediates 75 kDa suppression of ATM activation through the MRN complex, we treated cells with H2O2, which activates ATM independently of the C D Control RskWTRskCA MRN complex (39). Cells overexpressing Rsk displayed no change Mre11 676P 85 kDa Mre11 2A in ATM activation following H2O2 treatment, in agreement with WT Mre11 Mre11 641AMre11 676AMre11 688A Flag 85 kDa our conclusion that Rsk silences ATM activation through suppres- Rsk2 + - +++ ++ D Mre11 85 kDa sion of MRN complex binding to DNA (Fig. S3 ). 100 kDa 32P Mre11 46 kDa 75 kDa S6P S676 on Mre11 is the Target of Rsk. To identify specific target(s) of 100 kDa Rsk, we isolated DNA-bound proteins from crude S egg extracts Loading: Mre11 and incubated the precipitates with Rsk and radiolabeled ATP. 75 kDa The one major protein band identified by SDS/PAGE and autoradiography was ∼85 kDa, which is the molecular weight of E Control PMA PMA+SL0101 Mre11, suggesting that Mre11 might be a direct target of Rsk Mre11 676P 85 kDa A fi (Fig. S4 ). This result was con rmed using His-tagged Mre11 85 kDa recombinant Mre11, which could be directly phosphorylated by 37 kDa Rsk2 kinase (Fig. 4A). To determine if the decreased MRN/ S6P dsDNA binding was cause by phosphorylation of Mre11, we prephosphorylated Mre11 with Rsk2 and then incubated Mre11 Fig. 4. Rsk phosphorylates Mre11 at S676. (A)Purified Mre11 protein was with crude S extracts for 15 min (to allow association of other treated with Rsk and radiolabeled ATP (in kinase buffer), and labeled proteins required factors) before precipitating the dsDNA. As shown in were subjected to SDS/PAGE and autoradiography. (B) His-tagged Mre11 protein Fig. 4B, prephosphorylation by Rsk2 reduced the binding of was phosphorylated in vitro using RSK kinase and added to Xenopus egg Mre11 to dsDNA, suggesting that phosphorylation of Mre11 extracts for 10 min. Biotin-tagged dsDNA was used to pull down Mre11, and samples were analyzed for Mre11/dsDNA interaction by His-immunoblotting. In contributes to Rsk-mediated inhibition of the DSB response. To one sample (Rsk*), SL0101 and phosphatase inhibitors were added to extracts to identify the relevant sites of phosphorylation on Mre11, we prevent further phosphorylation by Rsk or dephosphorylation by phosphatases. phosphorylated recombinant His-tagged Mre11 with Rsk in vitro fi (C) Equal amounts of His-tagged Mre11 protein and mutants were treated as in and analyzed the protein by mass spectrometry. We identi ed A.(D) Lysates from 293T cells transfected with WT or constitutively active (CA) four phosphorylated residues: 2, 641, 676, and 688. Although Rsk were analyzed for endogenous S676 phosphorylated Mre11 by immuno- none of those four sites was identical to the Rsk consensus site blotting with phospho-specific antibody. (E) Lysates from 293T cells treated with RXRXXpS/T, Rsk can target nonoptimal sites (40). We mutated PMA ± SL0101 were analyzed for endogenous S676 phosphorylated Mre11 by each of the identified sites and performed in vitro Rsk assays immunoblotting with phospho-specific antibody.

20608 | www.pnas.org/cgi/doi/10.1073/pnas.1306328110 Chen et al. Downloaded by guest on October 2, 2021 Fig. 5. Phosphorylation of Mre11 on S676 A Empty Mre11 WT Mre11 676A B Beads reduces its binding to DNA and reverts check- - - + - + Mos point inhibition. (A) Mre11 DNA-binding domains Nuclease Domain DBD-1 DBD-2 Mre11 75 kDa were within its C-terminal half. (B) Xenopus 75 kDa crude S extracts pretreated with MBP-Mos or KU70 MBP and supplemented with WT or mutant 2 641 676 688 75 kDa Mre11 proteins were incubated with biotin-tag- Input: Mre11 ged dsDNA-bound avidin beads. The amount of WTMre11 Mre11 protein bound to the beads was analyzed MBP Mos by immunoblotting. (C)WTormutantMre11 C Empty D WT Mre11 Mre11 676A Min 0 5 10 20 30 0 5 10 20 30 beads proteins pretreated with Rsk were incubated with Rsk + - + - + P-ATM 250 kDa biotin-tagged dsDNA-bound avidin beads in PBS IP: DNA buffer for 20 min. Samples were then processed 85 kDa WB: Mre11 ATM 250 kDa as in B.(D) Xenopus crude S extracts with Mre11 WT or S676A proteins treated with MBP-Mos or E Mre11 676A MBP Mos MBP and then dsDNA were immunoblotted for 80 G2/M * Min 0 5 10 20 30 0 5 10 20 30 percentage phosphorylated ATM. (E) 293T cells transfected % P-ATM with FLAG-tagged constitutively active Rsk2 and 250 kDa HA-tagged Mre11 S676A or Mre11 mutated to A

250 kDa at S/T 2, 641, and 688 were stained with propi- dium iodide, anti-HA, and anti-FLAG antibody. 40 DNA profiles of total and HA-positive/FLAG-posi- F Mre11 foci *** tive populations were analyzed. *P < 0.01. Error positive% * ** bars indicate SD. (F) HeLa cells infected with Mre11 0.9 shRNA and transfected with shRNA-resistant WT 0 Mre11 or the S676 mutant were treated with PMA and SL0101 and then with NCS. Fixed cells were Ctrl 0.6 Ctrl stained with DAPI and anti-Mre11 antibody and Mre11 676A only Mre11 3else+Rsk WT Mre11 Mre11 676A +Rsk Mre11 676A were analyzed by fluorescence microscopy (40×). No NCS NCS 0.3 * Representative images are shown in Fig. S6.Cells with more than five clear Mre11 foci were counted as Mre11-positive. The quantitation of more than 0 fi NCS NCS+PMA NCS+PMA+SL0101 50 cells from at least eight random elds from each of three experiments is shown. *P < 0.001; **P < 0.001; ***P < 0.001. Error bars indicate SD.

reduced the amount of DNA-bound Mre11, but this effect was not Our findings are distinct from these reports in demonstrating seen when the S676A mutant was used (Fig. 5C). that Rsk-mediated inhibition of ATM activation can be achieved Consistent with the role for Mre11 S676 phosphorylation in the by targeting steps upstream of ATM activation. In addition, Rsk control of ATM by Rsk, we found that the ability of Mos to inhibit can suppress G2/M checkpoint function following DSB in cells DSB-induced ATM activation in Xenopus egg extracts was damp- knocked down for Chk1, suggesting that Rsk phosphorylation ened significantly by the addition of the S676A mutant Mre11 (Fig. of Mre11 contributes to DSB-induced G2 checkpoint silencing 5D). Moreover, upon expression in 293T cells, the S676A mutant independently of Chk1 (Fig. S7A). conferred resistance to Rsk-mediated G2/M checkpoint inhibition, The finding that phosphorylation on Mre11 at S676 inhibited whereas the control mutant (mutated at the other sites originally its binding to the DSB also is consistent with a previous report seen by mass spectrometry, 2A/641A/688A) did not (Fig. 5E). We demonstrating that mutation of eight SQ/TQ sites on Mre11, further investigated whether Rsk directly inhibited the formation of including S676, undermined the negative feedback loop whereby Mre11/MRN foci in Mre11-mutant cells. We knocked down en- ATM terminates the checkpoint response by inhibiting DNA dogenous Mre11 in HeLa cells using shRNA and transfected cells binding by Mre11 (32). Our study strongly suggests that S676 is, with shRNA resistant WT Mre11 or S676A. PMA treatment re- at least in part, responsible for such inhibition and that S676 duced the Mre11 foci in cells expressing WT Mre11 but not in the phosphorylation can prevent binding of Mre11 to dsDNA not cells expressing the S676A mutant (Fig. 5F and Fig. S6). Moreover, only in Xenopus extracts but also in mammalian cells. In addi- SL0101 reversed the effect of PMA in the WT-expressing cells, as is tion, we implicate Mre11 phosphorylation as a factor in a critical consistent with a role of Rsk in controlling the formation of Mre11/ checkpoint inhibitory pathway that results from high ambient MRN foci via phosphorylation of Mre11 S676 (Fig. 5F and Fig. S6). MAPK signaling in cancer cells. Interestingly, Di Virgilio et al. CELL BIOLOGY suggested that ATM is not the only kinase responsible for the Discussion phosphorylation of the observed eight SQ/TQ phospho-sites We have shown here that Rsk signaling impairs cell-cycle arrest by (32). Indeed, it is possible that Rsk not only participates in the DSB-induced G2/M checkpoint by inhibiting the ATM pathway. a checkpoint inhibitory pathway but also acts downstream of This effect appears to involve a failure to activate ATM through ATM in the pathway of feedback inhibition. In other words, it MRN-mediated recruitment to sites of DNA damage. We found may be that ATM can inhibit Mre11 by activating Rsk. We note that Mre11 protein, a core constituent of the MRN complex, was that the converse cannot be true—i.e., Rsk cannot activate ATM to phosphorylated by Rsk both in vitro and in intact cells. Furthermore, phosphorylate Mre11—because ATM inhibition did not preclude through mutagenesis experiments, one of the identified phosphory- Rsk-mediated phosphorylation or inhibition of Mre11. lation sites, S676, was demonstrated to be essential for functionally The DNA-damage response and its regulation have been shown inhibiting Mre11. Together, these findings provide a mechanism for to play a critical role in tumorigenesis; the DSB-response pathway Rsk-induced inhibition of DSB checkpoint function. can be turned off early in tumor development in multiple types A recent paper investigating the role of Rsk in G2/M arrest of cancer. This abrogation of checkpoint function prevents cell- following doxorubicin treatment reported that Rsk could phos- cycle arrest, leads to error-prone DNA replication, and promotes phorylate Chk1 at S280, the site that is targeted by Akt kinase to accumulation of multiple cellular mutations (which can fuel can- inhibit Chk1 activity (30). However, a similar study showed that cer progression) (43, 44). Moreover, because the primary goal of the same phosphorylation on Chk1 had the opposite effect (31). radiation therapy is the generation of abundant DSBs to induce

Chen et al. PNAS | December 17, 2013 | vol. 110 | no. 51 | 20609 Downloaded by guest on October 2, 2021 permanent cell-cycle arrest and/or apoptosis, the overriding of inhibitors may extend the utility of both irradiation and DNA- DSB-induced checkpoint function can undermine the utility of this damaging chemotherapeutics. treatment modality. Indeed as shown in Fig. S7B,inthecellswe tested, up-regulation of Rsk rendered cells resistant to excessive Materials and Methods DSB-induced cell death. However, it should be noted that in some Detailed information on materials and protocols is given in SI Materials radioresistant tumors the DSB response and DNA repair are up- and Methods. regulated rather than inactivated, leading to efficient repair of radiation-induced DNA damage (45). Consequently, the ther- Xenopus Extracts and DNA Immunoprecipitation. Cytostatic factor (CSF) apeutic benefit of inhibiting or activating the DSB response is extracts and interphase extracts were prepared as previously described (38). likely to vary depending on tumor type. This variance has clear Detailed information is provided in SI Materials and Methods. implications for Rsk as a target of tumor therapy. In cancer cells that inhibit their checkpoint function through Rsk, Rsk inhibition Immunostaining. HeLa cells were grown on coverslips, were treated with dif- might be synergistic with ionizing radiation. ferent drugs as described in Results,andwerefixed with 4% (vol/vol) para- The consequences of inhibiting Rsk for cancer therapy extend formaldehyde for 20 min. Immunostaining was performed as described beyond the impact on cell-cycle checkpoints and DNA repair. previously (47) using anti-γH2AX antibody. Recently, our laboratory reported that Rsk can phosphorylate the apoptotic inducer Apaf-1 and thereby reduce its apoptotic ACKNOWLEDGMENTS. We thank John Blenis (Harvard Medical School) for activity (21). Similarly, it has been reported that Rsk can inhibit providing the Rsk plasmids; William Dunphy (California Institute of Technol- the action of Bax/Bad complexes, a pivotal mediator of cell death ogy) for the Xenopus ATM and Nbs1 antibody; Bin Li (Duke Cancer Institute Flow Core Facility) for FACS analyses; Jiyeon Kim for Rsk siRNA and scientific following both chemotherapy and radiation (46). Therefore, at advice; Denise Ribar, Wanli Tang, Manabu Kurokawa, Christopher Freel, and least for tumors with high Rsk activity, inhibition of Rsk may Stephanie Freel for technical assistance; and members of the S.K. laboratory restore DNA damage-induced cell-cycle arrest while at the same for helpful discussions. This work was supported by National Institutes of time removing the brake on apoptosis. In these tumors, Rsk Health Grants R01 GM080333 and CA102707 (to S.K.).

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